Optogenetic-mediated increases in in vivo spontaneous activity disrupt pool-specific but not dorsal-ventral motoneuron pathfinding

Abstract

Rhythmic waves of spontaneous electrical activity are widespread in the developing nervous systems of birds and mammals, and although many aspects of neural development are activity-dependent, it has been unclear if rhythmic waves are required for in vivo motor circuit development, including the proper targeting of motoneurons to muscles. We show here that electroporated channelrhodopsin-2 can be activated in ovo with light flashes to drive waves at precise intervals of approximately twice the control frequency in intact chicken embryos. Optical monitoring of associated axial movements ensured that the altered frequency was maintained. In embryos thus stimulated, motor axons correctly executed the binary dorsal-ventral pathfinding decision but failed to make the subsequent pool-specific decision to target to appropriate muscles. This observation, together with the previous demonstration that slowing the frequency by half perturbed dorsal-ventral but not pool-specific pathfinding, shows that modest changes in frequency differentially disrupt these two major pathfinding decisions. Thus, many drugs known to alter early rhythmic activity have the potential to impair normal motor circuit development, and given the conservation between mouse and avian spinal cords, our observations are likely relevant to mammals, where such studies would be difficult to carry out.

Keywords: axonal guidance; motoneuron development; spinal cord development; spontaneous neural activity.

Fig. 1.

Light activation of ChR2 increases bursting frequency and results in segmental misprojections and altered axonal fasciculation. (A) Schematic showing the D-V and subsequent pool-specific pathfinding decisions made by sartorius (dark green), femorotibialis (light green), and adductor (red) motoneurons at base of limb. (B) Bar graph showing intervals between episodes of axial movements in controls and embryos chronically activated by light between St 20–26 (n = 4) or St 20–St 30/31 (n = 4) (mean ± SEM). (C–H) Transverse limb sections showing axons orthogradely labeled from LS1 with Di-Asp (green) and LS2 with Di-I (red) in control and stimulated embryos. (C and D) At D-V, choice point gray and white arrowheads indicate dorsal and ventral trunks, respectively. White arrows in C show intermingling of LS1 and LS2 axons in control. (E and F) Axons from LS1 and LS2 in control intermingle extensively in the ventral obturator nerve trunk but remain as separate fascicles in the experimental embryo. (G) More distally, both LS1 and LS2 axons contribute to the femorotibialis nerves and became extensively intermingled as shown by colocalization of red and green dye (yellow) in controls. (H) After treatment in some embryos, as in this case, only LS2, contributed. (C′, D′, E′, F′, G′, and H′) Same sections as shown in C, D, E, F, G, and H immunostained with NCAM antibody to reveal the entire nerve pattern. In C–H, dorsal is up, anterior is right. (Scale bars, 50 μm.)

Fig. 2.

The location of HRP⁺ and Er81⁺ motoneuron somas after retrograde labeling from St 30–31 sartorius or femorotibialis muscles in controls and light-activated (experimental) embryos. Motoneurons retrogradely labeled with HRP from the sartorius in control (B) and experimental embryo (D). Thin dotted line denotes the normal sartorius motoneuron pool boundary at LS1. (A and C) Same sections showing Er81 expression. Arrows (C and D) show misplaced motoneuron expressing Er81. (F and H) Motoneurons labeled from femorotibialis in control and experimental embryo. Thin dotted line denotes normal femorotibialis pool boundary at LS2. Arrows in H show misplaced motoneurons. (E and G) Same sections showing that these misplaced motoneurons do not express Er81. (Scale bar, 30 µm.) In A–H, the thick dotted line shows lateral edge of gray matter; dorsal is up, lateral is right. (Right) Schematic shows sartorius and femorotibialis pool locations at LS1 and LS2. (Bottom) Left and Center bar graphs show proportion of sartorius or femorotibilais motoneurons, respectively, that wrongly projected to the other muscle in controls and embryos activated at twice the normal frequency (n = 3, *P < 0.05 for both). Right bar graph shows proportion of motoneurons retrogradely labeled from sartorius that were Er81-positive (n = 3, *P < 0.05).

Fig. 3.

Segmental distribution of motoneurons properly (white bars) or improperly (black bars) projecting to the sartorius and the femorotibialis muscles in control and light-activated embryos. Histograms of the rostrocaudal locations of motoneuron somas in cross-sections of the spinal cord after retrograde labeling with HRP from the control sartorius (A) or femorotibialis (B) muscles as a percentage of the total HRP labeled cells. In controls, all motoneurons were located in the appropriate pool position within the spinal cord (n = 3 in A; n = 4 in B). (C and D) In treated embryos a proportion of motoneurons (black bars) in LS1 and LS2 were found in inappropriate pool locations for the sartorius (n = 3) and the femorotibialis (n = 4).

Fig. 4.

Electromyograms of bursting patterns from sartorius and femorotibialis muscles in control and light treated embryos. (A) Traces of two consecutive bursts within a bursting episode recorded simultaneously from the sartorius (Upper) and the femorotibialis (Lower) muscles of a stage 33.5 control embryo. The bracket above the first burst indicates one cycle of activity. The sartorius inhibitory period is shown by the black bars. (Scale bar, 1 s.) (B and C) A single burst from a control (B) and treated (C) embryo. Arrow and arrowheads indicate motoneurons firing at inappropriate times in the experimental sartorius and femorotibialis, respectively. (D–G) Histograms of traces from two St 34 embryos, a control (D and F, n = 7) and a light-activated embryo (E and G, n = 6) showing the probability of a muscle firing during the cycle preceding and subsequent to the 0 time point of a given cycle. Following treatment, motoneurons fire at inappropriate times during a cycle, consistent with motoneurons innervating wrong muscles (see text for further explanation). All six light-treated embryos examined exhibited motoneurons firing at inappropriate times whereas none of the five controls did.